systematics morphometric analysis of three putative...

SYSTEMATICS

Morphometric Analysis of Three Putative Species of Bemisiatabaci (Hemiptera: Aleyrodidae) Species Complex From India

RAHUL CHAUBEY,1,2,3 R. J. ANDREW,3 N. C. NAVEEN,1 R. RAJAGOPAL,4 BILAL AHMAD,5 AND

V. V. RAMAMURTHY1

Ann. Entomol. Soc. Am. 1–13 (2015); DOI: 10.1093/aesa/sav028

ABSTRACT The Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a pest of agricultural andhorticultural crops. The B. tabaci species complex consists of 36 morphologically indistinguishable puta-tive species. This study evaluates the morphometric variations of developmental stages, puparia, andadults in three putative species of B. tabaci occurring in India. The genetic identity of these studied pop-ulations were confirmed by mtCO1 analysis and revealed that the population from Amravati, Ludhiana,and Delhi were clustered with Asia I, Asia II1, and Asia II7 putative species, respectively. The morpho-logical comparisons showed that fourth instar and adult of Asia-II1 was comparatively larger than Asia-Iand Asia-II7. The positioning of sensorial cone on antennal segment 7 is much apart, and away from thesensorium in Asia-II1 while these are comparatively adjacent in Asia-I and Asia-II7 for both the sexes.The multivariate statistical analyses reveal that 31 measurements in puparia, 23 of male and 22 of femaleshow significant variations (P� 0.01). This was supported by scatter graphs derived from principal com-ponents and canonical discriminant analysis (CDA), and separate clustering was obtained for Asia-I,Asia-II1, and Asia-II7. Overall 91 and 99% of the classifications were correctly attributed by CDA forpuparia and adults which confirmed the distinction of these groups. The characters brought out in thisstudy could be used as a population/putative species specific markers in B. tabaci species complex andthese variations might enable distinguishing the other genetic groups too.

KEY WORDS Bemisia tabaci, developmental stage, puparia, adult, morphometric

Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae)is a haplodiploid, sap feeding arthropod pest in fieldcrops of warm to hot climates between 30� N and S ofthe equator. It is polyphagous and reported feeding onmore than 900 hosts (Hsieh et al. 2006). In the last twodecades, it is becoming a serious pest in short-livedherbaceous hosts including numerous agriculturaldicotyledon, horticultural and commodity crops (Brownet al. 1995). Phylogenetic studies by De Barro et al.(2011) suggested that B. tabaci is a cryptic species com-plex consisting of 24 morphologically indistinguishablespecies in 11 well-defined high-level groups. Thesewere later concluded as belonging to 36 putative spe-cies comparing their differences in genetic structure,host plant preferences, bacterial symbionts (Hu et al.2011; Boykin et al. 2012, 2013; Liu et al. 2012; Tayet al. 2012; Boykin 2013, 2014; Lee et al. 2013; Wanget al. 2013; Ashfaq et al. 2014; Boykin and De Barro2014) and interbreeding capabilities (Wang et al. 2010,Xu et al. 2010).

In India B. tabaci was first reported from cottonfields of Punjab in 1905 (Misra and Lambda 1929).The presence of invasive biotype B was reported fromthe field of Kolar and Bangalore (Banks et al. 2001).Later the Asian B. tabaci was differentiated into Asia I,Asia II, Asia II1, China, and MEAM 1 (Boykin et al.2007, Ahmed et al. 2011). Chowda Reddy et al. (2012)further resolved these and showed that the Asia I, AsiaII1, Asia II5, Asia II7, Asia II8, and MEAM1 putativespecies are present in the Indian subcontinent andAsia.

At present, biochemical and molecular biologicaltechniques are very helpful for differentiation of theseputative species within the species complex as wellas for identification of some morphologically indistin-guishable species or for construction of phylogenetictrees (Cervera et al. 2000, Calvert et al. 2001, Khasdanet al. 2005, Hsieh et al. 2007, Gueguen et al. 2009,McKenzie et al. 2009, Papayiannis et al. 2009, Dinsdaleet al. 2010, Sun et al. 2011). However, in terms oftaxonomy or systematics, morphology is considered theforemost basis for species separation (Gill andBrown 2010), not only due to the relationship be-tween morphological characteristics and phylogeny, butdue to convenience for identification as well (Yan2001). Therefore, it is necessary to conduct thor-ough investigations of B. tabaci species complexfor their morphological characters (Gill and Brown2010).

1 NAIP Subproject, C-3006 (Component-4), Division of Entomol-ogy, Indian Agricultural Research Institute, New Delhi 110012, India.

2 Corresponding author, email: [email protected] Department of Zoology, Hislop College, Nagpur 440001, Maha-

rashtra, India.4 Department of Zoology, University of Delhi, Delhi 11007, India.5 NAIP, PIU KAB-II, New Delhi 110012, India.

VC The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America.All rights reserved. For Permissions, please email: [email protected]

Annals of the Entomological Society of America Advance Access published April 28, 2015

In the 1990s morphology and morphometrics ofB. tabaci were extensively studied due to the invasionof B. tabaci B biotype to the United States. The B bio-type was described as silver leaf whitefly Bemisiaargentifolii based on morphological and allozymic char-acters (Bellows et al. 1994). And then more charactersof fourth-instar nymph (the pupal case) were found,such as absence of the fourth anterior margin setalpair, width of the thoracic tracheal folds, and width ofwax extrusions from the folds that could be used to sep-arate biotype B from A. However, the subsequent com-parison across 5 of the 11 major groups (Asia I, Asia II,Africa, Middle East Asia Minor, New World) showedthat the morphological characters previously used toseparate A from B were not useful for the reliable sepa-ration of any of the groups (Rosell et al. 1997, Calvertet al. 2001, Liu et al. 2012). As then, there have beenfew morphological studies on the B. tabaci speciescomplex. Gill and Brown (2010) examined the speciesstatus and the morphological variations in pupariawithin Bemisia and the relatives and concluded thatthese biotypes/putative species are a complex of crypticspecies which have evolved over time in isolation prob-ably without the need to change morphologically, butthey suggested conducting more thorough morphologi-cal studies at least in the puparia and adults.

Tay et al. (2012) stated that Mediterranean is thereal B. tabaci and illustrated the puparium and anten-nae of male and female, genitalia of male and variationin the cement gland of three adult female collected byGennadius. Li et al. (2013) conducted a thorough studyon the morphological characters and morphometrics ofsix biotypes from China and reported that lengthof operculum, length and width of lingula, andlength of antennal segments can be used to distinguishthese. This study also suggested that such results to beapplied to more biotypes from more plants andmore locations to test their stability and reliability.Hence, this study focusing on the morphology of devel-opmental stages, and morphometrics of puparia andadults of three putative species viz., Asia-I, Asia-II1and Asia-II7 of the B. tabaci species complex fromIndia.

Materials and Methods

Populations of B. tabaci were collected from differ-ent locations (Amravati, N 21� 010 60400 E 77� 550 00400,367 m; Ludhiana, N 30� 530 93200 E 75� 480 22900,240 m, and Delhi, N 28� 380 28.100 E 77� 100 12.200,213 m asl) in various agroclimatic zones of India fromtheir host plants viz., cotton (Gossypium spp.; Malvales:Malvaceae) and leucaena (Leucaena spp.; Fabales:Fabaceae). These were reared up to six generations oncotton in the insect proof climate control chamber atthe Indian Agricultural Research Institute, New Delhi,India, under controlled conditions of temperature(28 6 2�C), humidity (60 6 5%) and photoperiods(10 L� 14D). The samples drawn from these cultureswere subjected to morphometric studies after therequired molecular studies to confirm their geneticgroups.

Genomic DNA was extracted from single B. tabaciadult using DNeasy blood and tissue kit (QiagenGmbH, Hilden, Germany) according to manufacturer’sprotocol. The mtCOI region was amplified using theuniversal described by Frohlich et al. (1999) and Simonet al. (1994). All PCR analyses were conducted withTaq polymerase (Invitrogen, Sao Paulo, Brazil) in aPTC-200 thermocycler (Biorad, Germany). These am-plified PCR products were purified and sequenced inDNA analyzer (Scigenomics, Cochin, India).

The mtCO1 sequence obtained from each popula-tion was subjected to homology search using BasicLocal Alignment Search Tool (nBLAST) algorithm (Alt-schul et al. 1997, Schaffer et al. 2001) at NCBI (http://www.ncbi.nlm.nih.gov, last accessed 2 February 2014).The haplotypes of the consensus sequences of the allputative species identified in the previous reviews wereretrieved from GenBank (Chowda Reddy et al. 2012,De Barro and Boykin 2013, Ashfaq et al. 2014, Boykinand De Barro 2014) and aligned using the MUSCLEprogramme with default parameters (Edgar 2004).

Phylogenetic trees were constructed by using theGTRþ IþG (General Time Reversible model with aproportion of invariable sites and a gamma shaped dis-tribution of rates across sites) DNA substitution whichshowed the best Bayesian information criterion score(Posada 2008) and graphically displayed in a maximumlikely hood tree by using the program MEGA 6(Tamura et al. 2013). To assess the phylogenetic sup-ports for groupings on the tree, we performed a boot-strap resembling analysis (1,000 replication). The pairwise distances were calculated using the Kimura-2parameter model.

Developmental stages were identified according(Malumphy et al. 2009, Chaubey et al. 2010); measure-ments and photographs were taken in Leica M205FAstereozoom microscope attached with DFC425 digitalcamera at 350� to 1,600� (n¼ 30). Puparia and adultswere processed for mounting as recommended earlier(Mound 1963, Mohanty and Basu 1986, Chaubey et al.2010). The mounted specimens (n¼ 30) were observedunder the Leica DM100 phase contrast research mi-croscope at 400� for studying the essential characters.Photographs were taken on Leica DM500 stereozoommicroscope attached with DFC290 digital camera.

Adult antennal sensilla were subjected to analysis inscanning electron microscope (SEM). Specimen prepa-rations were adopted from Calvert et al. (2001). Theadults were dehydrated in a series of 70%, 90% andabsolute ethyl alcohol for 20 min in each and treated incarbolxylene for 2–4 h to remove the microscopic waxparticle. After that specimens were transferred to abso-lute ethyl alcohol for 10 min and then to hexamethyldisilazane (chemical dryer) for 2–3 min. SEM studieswere done with Zeiss EVOMA10 SEM at 20 Kv/EHTand at 3.20–6.40 kx after 24 nm palladium coating.A sample size of n¼ 30 for male and female from eachputative species was used and measurements were car-ried out by Smart Tiff V1.0.0.12 software.

Univariate one way single factor ANOVA was per-formed individually for all characters to select the sig-nificant characters as a prelude to identifying the

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significant ones (Kalaisekar et al. 2012). After this thepattern of clustering was analyzed using multivariatestatistical approaches (Tabachnick and Fidell 2007);Principal Component Analysis (PCA; SAS procedure;PRINCOMP; SAS version 9.1.3, SAS Institute Inc.,Cary, NC, USA) was used without any prior assumptionof groupings which assesses the components for totalvariation among the specimens by calculating linearcombination of variables that explain the maximum oftotal variation. Canonical Discriminant Analysis (CDA;SAS procedure; CANDISC) was used for calculatingthe linear combinations of variables that maximize theseparation of means of previously defined classes. Con-tribution of the variables best summarizing the differ-ences between classes is revealed by this technique. As,Discriminant Function Analysis (DFA; SAS procedure;DISCRIM) maximizes the variation among groups, itwas used to separate putative species of B. tabaci basedon morphometric data.

Results

Assigning of Populations to GeneticGroups. The global phylogenetic analysis with the par-tial mtCO1 sequences revealed that these are assign-able to the Asia I, and Asia II groups namely Asia II1and Asia II7. The Amravati (KF298442) populationsclustered with Asia I, while those from Ludhiana(KF298443) and Delhi (JQ023501) clustered with AsiaII1 and Asia II7, respectively (Fig. 1). The pair wisedistance between the putative species Asia II1 versusAsia II7 were found to be 0.098 while in Asia I versusAsia II1and Asia II7 it was 0.156 and 0.137,respectively.

Morphometrics.Developmental Stages. Morphology of developmen-

tal stages was studied especially the pattern and size ofwax fringe in the instars (Fig. 2). Morphometrics ofdevelopmental stages is shown in Table 1 which revealsthat the Asia I is comparatively smaller than the AsiaII1 and Asia II7 except for the length of fourth instarwhich was larger in Asia II1 followed by Asia I andAsia II 7. The breadth of wax fringe in first(0.028 6 0.008, 0.021 6 0.001, and 0.017 6 0.001) andin second instars (0.19 6 0.006, 0.016 6 0.001, and0.015 6 0.005) was more in Asia II7 as compared withAsia I and Asia II1, respectively. The length of anteriorwax fringe in fourth instar was comparatively more inAsia 1 (0.120 6 0.002) followed by Asia II1(0.091 6 0.002) and Asia II7 (0.063 6 0.003), while thatof posterior wax fringe was more in Asia II7(0.080 6 0.001) than Asia I (0.072 6 0.006) and Asia II1(0.069 6 0.001). All these measurements of develop-mental stages and their wax fringe vary and are statisti-cally significant at P� 0.001 except length of fourthinstar which is significant at P� 0.05.

Puparia. The morphometrics measurement ofimportant characters of puparia, and their significanceare listed in Table 2. The puparia from Asia II1 waslonger and wider than the Asia I and Asia II7. Thelength of tracheal fold (0.045) was longer while breadth

(0.104) was shorter in Asia II7. The vasiform orificewas shorter (0.068) in Asia II7 while it was broader(0.06) in Asia I; and a longer and wider operculum and

Fig. 1. Phylogenetic tree of mtCO1 gene sequences ofthe studied putative species of B. tabaci species complex(representative tree). Numbers above the branches indicatebootstrap value.

2015 CHAUBEY ET AL.: MORPHOMETRICS OF B. tabaci SPECIES COMPLEX 3

Fig. 2. Developmental stages of B. tabaci showing the wax fringe pattern; A, first instar; B, second instar; C, third instar;D, fourth instar.

Table 1. Morphometric measurements of developmental stages and there wax fringe Asia-I, Asia-II1, and Asia-II7 genetic group of B.tabaci (mean 6 SE mm; n¼30)

Characters Amravati Ludhiana DelhiAsia I Asia I-1 Asia II-7

First instar (L�B) 0.212 6 0.003� 0.130 6 0.002 0.264 6 0.003� 0.136 6 0.002 0.232 6 0.005� 0.144 6 0.003Wax fringe breadth 0.021 6 0.000 0.017 6 0.000 0.028 6 0.008Second instar (L�B) 0.314 6 0.003� 0.199 6 0.002 0.325 6 0.004� 0.182 6 0.002 0.333 6 0.007� 0.214 6 0.006Wax fringe breadth 0.016 6 0.000 0.015 6 0.005 0.019 6 0.006Third instar (L�B) 0.423 6 0.003� 0.263 6 0.004 0.442 6 0.004� 0.258 6 0.003 0.454 6 0.006� 0.322 6 0.009Fourth instar (L�B)* 0.687 6 0.004� 0.481 6 0.011 0.725 6 0.011� 0.495 6 0.012 0.665 6 0.009� 0.438 6 0.009Anterior wax fringe (L�B) 0.120 6 0.002� 0.022 6 0.000 0.091 6 0.002� 0.021 6 0.006 0.063 6 0.003� 0.022 6 0.002Posterior wax fringe (L�B) 0.072 6 0.001� 0.016 6 0.004 0.069 6 0.006� 0.017 6 0.004 0.080 6 0.001� 0.021 6 0.005

All the measurements were statistically significant at P< 0.001 and *was significant at P< 0.05.

Table 2. Statistically significant characters of B. tabaci puparia from Asia-I, Asia-II1, and Asia-II7 genetic groups (mean 6 SE mm;n¼30) in which P<0.01

Sr. No. Characters Amravati Ludhiana Delhi F-value P-valueAsia-I Asia II-1 Asia II-7

1 Length of the dorsal seta 3 0.126 6 0.008 0.130 6 0.004 0.014 6 0.010 73.008 9.03� 10-342 Length of the dorsal seta 1 0.132 6 0.010 0.138 6 0.005 0.019 6 0.009 64.769 2.55� 10-313 Length of the dorsal seta 5 0.122 6 0.006 0.075 6 0.000 0.009 6 0.011 38.861 4.44� 10-224 Length of the antennae 0.061 6 0.000 0.058 6 0.000 0.051 6 0.000 38.428 6.71� 10-225 Breadth of the vasiform orifice 0.060 6 0.004 0.057 6 0.006 0.052 6 0.006 36.485 4.42� 10-216 Length of the dorsal seta 2 0.117 6 0.006 0.087 6 0.000 0.007 6 0.012 35.141 1.68� 10-207 Length of the dorsal seta 6 0.052 6 0.001 0.005 6 0.002 0.006 6 0.008 28.138 2.67� 10-178 Length of caudal furrow 0.058 6 0.007 0.058 6 0.001 0.048 6 0.001 24.669 1.37� 10-159 Breadth of operculum 0.049 6 0.004 0.047 6 0.005 0.043 6 0.005 24.427 1.82� 10-1510 Length of the posterior submarginal setae 5 0.048 6 0.000 0.005 6 0.000 0.005 6 0.000 22.818 1.23� 10-1411 Length of the operculum 0.037 6 0.002 0.036 6 0.003 0.033 6 0.009 22.794 1.26� 10-1412 Length of the dorsal seta 4 0.059 6 0.012 0 0 21.897 3.74� 10-1413 Breadth of the pupal case 0.576 6 0.005 0.549 6 0.008 0.515 6 0.008 19.252 1.01� 10-1214 Length of first abdominal segment 0.043 6 0.001 0.044 6 0.001 0.042 6 0.000 18.506 2.63� 10-1215 Length of eighth abdominal segment from pocket to

caudal pore0.193 6 0.001 0.189 6 0.003 0.169 6 0.002 18.142 4.22� 10-12

16 Distance of vasiform orifice from anterior end 0.618 6 0.004 0.609 6 0.008 0.598 6 0.007 16.935 2.05� 10-1117 Length of the lingula 0.055 6 0.004 0.052 6 0.001 0.047 6 0.003 14.525 5.43� 10-1018 Distance of vasiform orifice from dorsal seta 6 0.021 6 0.002 0.018 6 0.004 0.016 6 0.007 14.243 7.89� 10-1019 Breadth of the tracheal fold 0.106 6 0.001 0.106 6 0.002 0.104 6 0.003 13.997 1.11� 10-0920 Length of the pupal case 0.796 6 0.005 0.744 6 0.011 0.720 6 0.009 12.842 5.65� 10-0921 Distance of transverse moulting suture from anterior end 0.376 6 0.002 0.351 6 0.004 0.348 6 0.004 12.526 8.86� 10-0922 Distance of transverse moulting suture from posterior end 0.388 6 0.003 0.394 6 0.007 0.373 6 0.006 10.495 1.7� 10-0723 Length of si� th abdominal segment 0.044 6 0.000 0.043 6 0.002 0.042 6 0.000 8.941 1.76� 10-0624 Length of the vasiform orifice 0.073 6 0.003 0.073 6 0.009 0.068 6 0.003 6.518 7.48� 10-0525 Distance between caudal seta 0.045 6 0.006 0.045 6 0.007 0.041 6 0.007 6.343 9.87� 10-0526 Distance between posterior submarginal seta 1 from

base of caudal seta0.043 6 0.000 0.044 6 0.000 0.040 6 0.000 6.074 1.5� 10-04

27 Distance between the dorsal seta 5 0.126 6 0.002 0.128 6 0.002 0.127 6 0.002 5.6611 2.91� 10-0428 Distance between ventral seta 0.156 6 0.006 0.146 6 0.006 0.127 6 0.008 4.556 1.7� 10-0329 Distance between dorsal seta 1 and base of proleg 0.144 6 0.001 0.146 6 0.002 0.140 6 0.003 4.1418 3.3� 10-0330 Length of the tracheal fold 0.043 6 0.001 0.038 6 0.001 0.045 6 0.000 3.496 9.3� 10-0331 Length of the ventral seta 0.032 6 0.004 0.027 6 0.007 0.018 6 0.002 3.029 9.52� 10-02


lingula were found in Asia I as compared with those ofAsia II1 and Asia II7.

The evaluation of morphometrics of puparia throughunivariate single factor one-way ANOVA revealed that31 of them had statistically significant variations atP� 0.01 (Table 2). These 31 characters when subjectedto multivariate analysis, it was observed that the firstfour Principal Components (PCs) with an eigen values>1 account for 76.8% of the total variation obtainedthrough PCA (Table 3), in which characters with maxi-mum loadings were considered as the major sources ofvariation. The plot for PC1 and PC2 shown in Figure 3brings out the grouping of these populations. Separateclustering was observed for Asia II7 with slight overlap-ping between Asia I and Asia II1. CDA was carried outwith prior grouping and using the populations as classi-fication variables. The projection of morphometric dataonto the first two canonical discriminant axes is shownin Figure 4. This clustering confirmed the groupingbrought out by the PCA. The first canonical root clearlydiscriminates the groups with major contribution beingfrom the length of operculum, breadth of vasiformorifice, breadth of operculum, and length of caudal fur-row; the second canonical root is able to discriminatedue to the contribution from length of abdominal seg-ment 1, length of vasiform orifice, length of caudal fur-row, breadth of vasiform orifice and distance ofvasiform orifice from base of dorsal setae 6 (Table 3).Overall 91% of the classifications were correctly attrib-uted and the result of cross validation accurately

classifies 93.3% of Asia II1 followed by 90% of those ofAsia I, and 90% of Asia II7 (Table 8).

Adults. The morphometrics of male and femaleshown in Tables 4 and 5 reveal that, in males bodylength was significantly varying at P� 0.01 between thethree putative species and it was comparatively largerin Asia I (1.0) than Asia II7 (0.997) and Asia II1(0.951). The length of antennae was also significantlyvarying between the three putative species, and it was

Fig. 3. PC coordinate of B. tabaci puparia from Asia-I,Asia-II1, and Asia-II7 genetic groups based on the analysisof 31 morphological variables onto first and second principalaxis.

Table 3. Contributions of variable coefficients of first four eigenvectors for PC analyses from measurement of B. tabaci puparia ofAsia-I, Asia-II1, and Asia-II7 genetic groups with its significance and total sample standardized canonical coefficients for CDA

Proportion of total variation Prin1 45.3% Prin2 21.3% Prin3 6.5% Prin4 3.5% Can1 Can2

PCL 0.242 � 0.129 0.095 � 0.065 �26.385 �44.197PCB 0.244 �0.093 0.056 0.090 7.860 18.147DTMSAM 0.198 �0.097 0.171 �0.115 �0.131 43.250DTMSPM 0.231 �0.133 0.026 �0.054 �23.209 �2.214DVOAM 0.231 �0.141 0.140 �0.046 17.635 34.388LABS1 0.183 �0.114 0.022 �0.172 �72.612 �145.603LABS6 0.204 �0.131 0.157 0.037 29.027 32.004L8thABPC 0.261 0.008 �0.034 0.017 �23.977 85.963TFL 0.008 �0.078 0.402 0.588 �15.150 42.952TFB 0.192 �0.177 0.095 0.040 �11.580 �46.325AL 0.175 0.180 �0.150 �0.018 77.517 �37.364LVO 0.243 �0.071 �0.005 �0.043 �70.202 �129.830LO 0.242 0.050 �0.094 �0.003 360.730 196.960LL 0.232 0.004 �0.033 0.103 �60.391 �20.004BVO 0.227 0.024 �0.056 0.162 249.277 237.401BO 0.234 0.033 �0.067 0.158 237.043 �13.422LCF 0.245 0.029 �0.068 �0.007 218.887 �152.297DCS 0.176 0.073 �0.158 0.097 �79.228 44.742LDS1 0.117 0.257 �0.302 0.035 14.733 �12.336DDS1PL 0.159 �0.069 �0.138 �0.200 �17.346 26.458LDS2 0.064 0.313 �0.108 0.128 �2.416 �21.593LDS3 0.109 0.280 �0.290 0.102 �5.004 �13.195LDS4 0.058 0.290 0.369 �0.150 0.868 �2.831LDS5 0.074 0.306 �0.097 0.141 10.625 28.368DDS5 0.122 �0.225 0.043 0.229 �27.359 �39.586LDS6 0.069 0.291 0.373 �0.133 �16.520 17.746LDS7 0.058 0.303 0.353 �0.132 7.331 30.694DVODS7 0.135 0.267 0.189 �0.124 51.723 �191.974LVS 0.098 0.265 0.040 0.040 7.621 �26.988DVS 0.105 �0.118 �0.063 �0.544 �2.052 �81.607DPSMS1CS 0.172 �0.075 �0.113 0.079 �26.397 �91.943P-value <0.001 <0.001 <0.001 <0.001


larger in Asia II1 (0.224) and shorter in Asia II7(0.174). The length of sensorial cone on antennal seg-ment (LSCAS) 3, 6, and 7 and distance of sensorialcone on 5 and 7 were significantly varying and LSCAS3 was larger in Asia II1, and LSCAS 6 and 7 wassmaller Asia II7 (Table 4). For females as in males, sizewas found to be significantly varying at P� 0.01 and itwas larger in Asia I (1.094) as compared to Asia II1(0.974) and Asia II7 (1.050). Also as regards length ofantennae, LSCAS 3, operculum and lingula and posi-tioning of sensorial cone on antennal segment 7 thiswas true. Critical observations revealed that the posi-tioning of sensorial cone on antennal segment 7 isbelow the sensorium in Asia II1, while it is adjacent tothe sensorium in Asia I and Asia II7 (Fig. 5) for boththe sexes.

The PCA and CDA. The morphometric evaluationof adults through univariate single factor one-wayANOVA revealed that of the measurements/observa-tions explored, 23 of male and 22 of female, had statis-tically significant variations at P� 0.01 (Tables 4 and 5).These significant characters were subjected to PCA,and the first five PCs with an eigen values >1 wereobserved to account for 74.2% in male and 77.1% infemale, of the total variation (Tables 6 and 7). The scat-ter plot for the PC1 and PC2 shown in Figures 6(male) and 7 (female) brings out the grouping of theseputative species. Separate clustering was observed forAsia II7 with slight overlapping between Asia I andAsia II1. The projection of CDA groups onto the firsttwo canonical discriminant axes is shown in Figures 8

(male) and 9 (female). This clustering obtained fromCDA confirmed the grouping brought out by PCA.The first canonical root clearly discriminates the groupswith major contribution being from LSCAS 6, 7, and 3,antennal segment 6 and distance of sensorium frombase of antennal segment 5 (in male); and also withregard to LSCAS 3, antennal segment 4, and lingula (infemale). The second canonical root is able to discrimi-nate from spacing between wax plate 2 and 3, LSCAS3 and distance of sensorial cone on antennal segment 6(for male), and LSCAS 7, antennal segment 4 and 1,and distance of sensorium on antennal segment (forfemale) (Tables 6 and 7). A cross validation of groupmembership was performed. The result of cross valida-tion accurately classifies Asia II1 and Asia II7 popula-tions (100%) followed by Asia I (96.67%) (Table 8).

Discussion

The B. tabaci species complex consists of multiplehaplotypes and number of well characterized behavior-ally differing variants (Brown et al. 1995, Perring2001). For the separation of putative species of theB. tabaci species complex ultimately molecular datahad been in vogue so far. The present analyses haveused the >3.5% divergence limits observed in mtCO1gene for identifying the genetic groups/putative species(Dinsdale et al. 2010, De Barro et al. 2011). The inter-pretations utilizing the global phylogenetic analysisreveal that these populations cluster with Asia I (Amra-vati), Asia II1 (Ludhiana), and Asia II7 (Delhi),

Fig. 4. CDA showing the coordinate of B. tabaci puparia from Asia-I, Asia-II1, and Asia-II7 genetic groups based on theanalysis of 31 morphological variables onto first and second canonical axis.


respectively. The identification of whiteflies depends onthe morphology of puparia and the taxonomic status ofB. tabaci has been problematic due to continuous mor-phological differences considered by taxonomists torepresent intraspecific variations (Russell 1957, Mound1963, Rosell et al. 1997). This study found that the sizeof developmental stages was larger in Asia I as com-pared with Asia II1 and Asia II7 except fourth instarwhich was longer in Asia II1. The wax fringe of first,second and fourth instars can be used as a reliablecharacter to differentiate the developmental stages of

these three putative species. The characteristic featureof wax fringe is used to identify species (Martin 1987)and biotypes/genetic groups/putative species of B.tabaci (Rosell et al. 1997, Yuan et al. 2003, Baoli et al.2009).

Extensive morphology and morphometrics studies ofB. tabaci showed that the morphological charactersused for separation of different biotypes/genetic groupswere not useful for their reliable separation (Rosellet al. 1997, Calvert et al. 2001, Liu et al. 2012). In thisstudy, of the characters chosen for morphometrics, 31

Table 4. Statistically significant characters of B. tabaci adults male from Asia-I, Asia-II1, and Asia-II7 genetic groups (mean 6 SEmm; n¼30) in which P<0.01

S. No. Characters Amravati Ludhiana Delhi F value P-valueAsia-I Asia-II1 Asia-II7

1 Spacing between wax plate 1 0.048 6 0.002 0.042 6 0.004 0.049 6 0.006 63.597 <0.0012 LSCAS 3 0.007 6 0.002 0.008 6 0.002 0.006 6 0.002 39.896 <0.0013 Length of antennae 0.217 6 0.024 0.224 6 0.030 0.174 6 0.037 38.564 <0.0014 Spacing between wax plate 3 0.041 6 0.001 0.041 6 0.001 0.046 6 0.001 27.235 <0.0015 LSCAS 7 0.010 6 0.001 0.010 6 0.002 0.008 6 0.002 27.212 <0.0016 Length of wax plate 1 0.049 6 0.001 0.045 6 0.000 0.045 6 0.001 26.394 <0.0017 Breadth of wax plate 1 0.098 6 0.001 0.092 6 0.001 0.089 6 0.001 22.368 <0.0018 Length of antennal segment 2 0.036 6 0.004 0.036 6 0.004 0.040 6 0.001 22.271 <0.0019 LSCAS 6 0.005 6 0.001 0.005 6 0.002 0.004 6 0.001 21.786 <0.00110 Breadth of wax plate 2 0.098 6 0.001 0.092 6 0.002 0.089 6 0.001 19.997 <0.00111 Length of clasper 0.094 6 0.001 0.092 6 0.000 0.097 6 0.001 19.609 <0.00112 Spacing between wax plate 2 0.043 6 0.001 0.042 6 0.002 0.046 6 0.003 15.442 <0.00113 Length of wax plate 3 0.041 6 0.000 0.039 6 0.001 0.044 6 0.002 14.981 <0.00114 Length of adeagus 0.086 6 0.001 0.091 6 0.001 0.092 6 0.002 14.655 <0.00115 Length of wax plate 2 0.043 6 0.001 0.040 6 0.001 0.045 6 0.001 13.797 <0.00116 Body Length 1.000 6 0.003 0.951 6 0.032 0.997 6 0.008 13.187 <0.00117 Length of lingula 0.028 6 0.001 0.027 6 0.000 0.025 6 0.000 12.875 <0.00118 Length of antennal segment 6 0.023 6 0.005 0.023 6 0.004 0.020 6 0.002 7.615 <0.00119 Distance of sensorial cone from base of antennal segment 6 0.016 6 0.004 0.016 6 0.003 0.014 6 0.005 7.347 <0.00120 Distance of sensorium from base of antennal segment 5 0.020 6 0.003 0.021 6 0.005 0.020 6 0.002 6.564 <0.00121 Breadth of clasper 0.027 6 0.001 0.028 6 0.000 0.026 6 0.001 6.315 <0.00122 Length of antennal segment 5 0.025 6 0.003 0.026 6 0.005 0.024 6 0.002 5.272 <0.00123 Spacing between wax plate 4 0.041 6 0.000 0.041 6 0.001 0.046 6 0.001 3.772 <0.001

Table 5. Statistically significant characters of B. tabaci adults female from Asia-I, Asia-II1, and Asia-II7 genetic groups (mean 6 SEmm; n¼30) in which P<0.01

S. No. Characters Amravati Ludhiana Delhi F value P-valueAsia-I Asia-II1 Asia-II7

1 LSCAS 6 0.004 6 0.001 0.005 6 0.00 0.004 6 0.001 89.865 <0.0012 Length of lingula 0.036 6 0.000 0.034 6 0.001 0.031 6 0.001 86.511 <0.0013 Length of gonapophyses 0.141 6 0.012 0.126 6 0.001 0.126 6 0.013 74.437 <0.0014 LSCAS 3 0.008 6 0.001 0.009 6 0.002 0.006 6 0.001 59.629 <0.0015 Body Length 1.094 6 0.009 0.974 6 0.011 1.050 6 0.010 38.184 <0.0016 Length of wax plate 2 0.073 6 0.000 0.063 6 0.001 0.062 6 0.001 38.149 <0.0017 Length of wax plate 1 0.073 6 0.001 0.065 6 0.001 0.064 6 0.001 32.479 <0.0018 Length of antennal segment 1 0.012 6 0.001 0.011 6 0.002 0.010 6 0.000 30.807 <0.0019 Length of antennae 0.237 6 0.023 0.251 6 0.026 0.197 6 0.023 25.368 <0.00110 Length of operculum 0.034 6 0.001 0.032 6 0.001 0.031 6 0.001 20.902 <0.00111 Breadth of gonapophyses 0.156 6 0.001 0.145 6 0.002 0.145 6 0.001 17.312 <0.00112 Length of cement gland 0.058 6 0.000 0.057 6 0.000 0.053 6 0.001 17.199 <0.00113 LSCAS 7 0.010 6 0.001 0.011 6 0.002 0.009 6 0.001 16.152 <0.00114 Distance of sensorium from base of antennal segment 5 0.022 6 0.003 0.024 6 0.004 0.023 6 0.003 13.041 <0.00115 Spacing between wax plate 1 0.064 6 0.001 0.058 6 0.002 0.067 6 0.002 12.945 <0.00116 Breadth of wax plate 2 0.121 6 0.001 0.110 6 0.002 0.110 6 0.002 11.708 <0.00117 Spacing between wax plate 2 0.063 6 0.001 0.058 6 0.001 0.066 6 0.002 10.969 <0.00118 Length of antennal segment 5 0.027 6 0.003 0.029 6 0.005 0.028 6 0.004 7.296 <0.00119 Breadth of wax plate 1 0.120 6 0.001 0.111 6 0.002 0.111 6 0.002 7.149 <0.00120 Length of antennal segment 2 0.039 6 0.008 0.042 6 0.004 0.040 6 0.001 6.028 <0.00121 Distance of sensorium from base of antennal segment 7 0.021 6 0.005 0.020 6 0.004 0.019 6 0.003 5.397 <0.00122 Length of antennal segment 4 0.019 6 0.004 0.017 6 0.002 0.018 6 0.002 5.144 <0.001


characters were found to vary significantly, and PCAand CDA confirmed their distinction as separate clus-tering was obtained for Asia I, Asia II1, and Asia II7.Amongst these, length of operculum, breadth of vasi-form orifice, breadth of operculum, and length of cau-dal furrow discriminate the putative species withmaximum contribution in canonical correlation. CDAwith a higher classification (93.3 and 90%) values pro-poses to establish the probable validity of studied mor-phological characters towards delineation of B. tabacispecies complex and its putative species. An observa-tion onto the morphology of puparia among the puta-tive species revealed that the length of vasiform orificeand tracheal fold was smaller in Asia II7 as comparedwith Asia II1 and Asia I. These results are in agreementwith those of Lozier et al. (2008), Jayasekera et al.(2010), and Thomas et al. (2011, 2014). It could be

observed that some previous conclusions of David andAnanthakrishnan (1976), Mohanty and Basu (1986),and Mound (1963) are also in agreement with thisstudy.

Adult morphology is neglected in whitefly taxonomy,probably due to less morphological differences (Moundand Halsey 1978). In this study we observed that bothmale and female of Asia I was longer as compared toAsia II1 and Asia II7. LSCAS s shows significant varia-tion among these putative species which is similar tothose observed by Calvert et al. (2001). In the morpho-metrics, 23 of male and 22 of female measurementswere found to be statistically significantly varying. Simi-larly PCA and CDA also revealed and confirmed thedistinction of these genetic groups as separate cluster-ing was obtained for Asia I, Asia II1, and Asia II7.Length of sensorial cone and distance of sensorium on

Fig. 5. Apical (seventh) segments of antennae of adult whitefly B. tabaci. A, female of Asia-II1; B, male of Asia-II1; C,female of Asia-I; D, male of Asia-I; E, female of Asia-II7; F, male of Asia-II7.


Table 6. Contributions of variable coefficients of first four eigenvectors for PC analyses from measurement of B. tabaci adult male ofAsia-I, Asia-II1, and Asia-II7 genetic groups with its significance and total sample standardized canonical coefficients for CDA

Proportion of total variation Prin1 Prin2 Prin3 Prin4 Prin5 Can1 Can229.6% 17.1% 10.9% 10.1% 6.5%

L 0.207 0.367 0.054 0.013 �0.026 4.430 17.505LWP1 0.036 0.377 �0.019 �0.180 0.205 467.484 �337.283BWP1 �0.050 0.465 �0.038 �0.070 �0.125 242.478 141.763SWP1 0.282 0.041 0.116 �0.151 0.321 �244.952 910.658LWP2 0.235 0.261 0.146 �0.001 0.040 �280.075 237.553BWP2 �0.041 0.456 �0.082 �0.076 �0.131 �320.386 �3.620SWP2 0.282 0.015 0.284 0.036 0.027 27.041 �425.970LWP3 0.279 0.163 0.224 0.125 0.105 �145.811 �3.651SWP3 0.312 �0.076 0.255 0.114 0.037 461.819 �472.239SWP4 0.166 �0.032 0.261 0.370 �0.023 �102.217 116.042LC 0.261 0.053 �0.083 0.156 0.124 �151.083 �70.557BC �0.074 0.060 0.029 0.352 �0.445 324.758 76.641LA 0.120 0.008 0.147 0.363 �0.338 �127.767 �165.548LL �0.113 0.348 0.045 �0.036 �0.110 343.611 �68.159AL �0.299 0.098 0.273 �0.054 �0.010 156.655 17.206LSC3 �0.261 0.102 0.055 0.352 0.053 521.554 �418.793LAS5 �0.117 �0.034 0.415 �0.317 �0.280 127.851 �113.309DSAS5 �0.144 �0.068 0.451 �0.252 �0.253 �668.608 104.951LAS6 �0.224 0.019 0.284 0.005 0.364 �537.787 383.060LSCAS6 �0.202 0.031 0.130 0.302 0.241 1,188.748 70.664DSCAS6 �0.228 0.010 0.281 0.012 0.299 279.845 �429.726LSCAS7 �0.255 0.099 0.021 0.304 0.203 �764.600 279.355LAS2 0.182 �0.174 0.171 �0.105 �0.018 �343.429 �73.777P-value <0.001 <0.001 <0.001 <0.001 <0.001

Table 7. Contributions of variable coefficients of first four eigenvectors for PC analyses from measurement of B. tabaci adult femaleof Asia-I, Asia-II1, and Asia-II7 genetic groups with its significance and total sample standardized canonical coefficients for CDA

Proportion of total variation Prin1 Prin2 Prin3 Prin4 Prin5 Can1 Can230.5% 21.9% 10.8% 7.9% 6.1%

L 0.306 �0.134 0.048 �0.139 0.060 �14.277 2.444LWP1 0.352 0.022 �0.011 0.059 0.031 15.180 �21.448BWP1 0.330 �0.058 0.065 0.207 0.046 6.863 �45.550SWP1 0.223 �0.241 0.291 0.171 �0.169 �73.325 43.858LWP2 0.358 0.039 0.020 0.018 0.004 69.904 139.050BWP2 0.349 �0.045 0.054 0.150 0.037 71.244 �2.822SWP2 0.200 �0.241 0.325 0.191 �0.193 �47.353 �99.518LO 0.295 0.044 �0.015 0.201 �0.184 95.237 276.437LL 0.216 0.265 �0.193 0.082 �0.101 302.747 60.135LCG 0.089 0.217 �0.168 0.006 �0.027 112.564 �0.241LGP 0.265 0.110 �0.111 �0.324 0.286 24.768 156.244BGP 0.219 0.093 �0.038 �0.315 0.391 �3.189 �46.902AL 0.051 0.399 �0.050 0.152 0.042 47.253 �12.494LSCAS3 �0.054 0.360 �0.011 0.141 �0.193 1,291.248 �144.993LAS4 0.061 0.121 0.349 �0.435 �0.076 �734.072 304.156LAS5 �0.108 0.185 0.499 0.025 0.228 107.280 91.459DSAS5 �0.115 0.149 0.478 0.117 0.313 182.074 �445.843LSCAS6 �0.051 0.352 0.005 0.323 �0.067 143.564 �82.879LSCAS7 0.009 0.325 0.109 �0.039 �0.302 76.933 �353.992DSAS7 0.072 0.261 0.299 �0.211 �0.187 �35.648 160.995LAS1 0.171 0.246 �0.131 �0.019 0.184 �110.586 390.224LAS2 �0.060 �0.008 0.002 0.444 0.536 �30.046 �59.106P-value <0.001 <0.001 <0.001 <0.001 <0.001


Fig. 6. PC coordinate of B. tabaci adult male from Asia-I,Asia-II1, and Asia-II7 genetic groups based on the analysis of23 morphological variables onto first and second principal axis.

Fig. 8. CDA showing the coordinate of B. tabaci adult male from Asia-I, Asia-II1, and Asia-II7 genetic groups based onthe analysis of 23 morphological variables onto first and second canonical axis.

Fig. 7. PC coordinate of B. tabaci adult female fromAsia-I, Asia-II1, and Asia-II7 genetic groups based on theanalysis of 22 morphological variables onto first and secondprincipal axis.

Table 8. Cross validation matrix of the DFA from measurement of B. tabaci puparia and adults of Asia-I, Asia-II1, and Asia-II7genetic groups

Number of observations and percent classified

Puparia Male Female

Population Amravati Delhi Ludhiana Amravati Delhi Ludhiana Amravati Delhi LudhianaAmravati 27 0 3 29 0 1 29 0 1

90.00 0 10.00 96.67 0 3.33 96.67 0 3.33Delhi 0 27 3 0 30 0 0 30 0

0 90.00 10.00 0 100 0 0 100 0Ludhiana 2 0 28 0 0 30 0 0 30

6.67 0 93.33 0 0 100 0 0 100Total 29 27 34 29 30 31 29 30 31

32.22 30.00 37.78 32.22 33.33 34.44 32.22 33.33 34.44


antennal segments in both male and female, and spac-ing between wax plates in female discriminate thegenetic groups with maximum canonical correlation.These findings are similar to and corroborate those ofLi et al. (2013). The positioning of sensorial cone onantennal segment 7 is below the sensorium in Asia II1,while it is more adjacent in Asia I and Asia II7, in boththe sexes. These explicit distinctions might provide sup-port for distinguishing the other putative species of B.tabaci too. However, a detailed study warranting moreputative species from various hosts and geographicallocations might be necessary to validate these findings.

Acknowledgments

We thank the National Agricultural Innovation Project(NAIP) of the Indian council for Agricultural Research(ICAR), India for funding this programme. Authors wantto thank Mahesh Rawat for providing assistance inPhotoshop.

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Received 14 August 2014; accepted 11 March 2015.


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